A system for studying basic biochemical and physiologic aspects of the contraction of intact mammalian smooth muscle has been further developed during the past year. Utilizing fresh strips of bovine trachealis muscle, methods have been refined to correlate the contractile response to certain agonists with the state of phosphorylation of myosin light and heavy chains, and myosin light chain kinase (MLCK). To determine whether force generation can be catalyzed by phosphorylation of myosin light chain (MLC) at amino acid residues other than the preferential MLCK site (serine 19), a phorbol ester activator of intracellular protein kinase C (PKC) and the physiologic peptide agonist Substance P have been used to cause contraction. In both cases the pattern of MLC phosphorylation qualitatively resembles that seen with other conventional methods of activation, with some variation in quantitative aspects. In addition, experiments have been undertaken to characterize biochemical correlates of tracheal relaxation, with particular interest in the response due to substances acting through cyclic AMP.